We have demonstrated the capacity of allogeneic or alloantigen-presenting immature dendritic cells (iDC), expressing low levels of major histocompatibility complex (MHC) class II and the co-stimulatory molecules, to prolong MHC-mismatched cardiac allograft survival when delivered before transplantation. This effect is potentiated by co-stimulation blockade, and the combination of iDC and co-stimulation blockade also reduced transplant vasculopathy. However, consistent indefinite graft survival via iDC administration is not observed, suggesting that infused iDC may be susceptible to maturation following exposure to endogenous inflammatory factors. Our recent data show that the 'tolerance-sparing' immunosuppressive drug rapamycin (RAPA) results in stably-immature dendritic cells (RAPA-DC) that are markedly impaired in their ability to stimulate T cells via direct and indirect pathways of allorecognition, even following exposure to potent inflammatory stimuli, RAPA-DC infusion, in the absence of any administered immunosuppression, induced indefinite cardiac allograft survival in 40% of animals. Further evidence suggests that RAPA-DC, when contrasted to iDC, selectively expand Foxp3+ CD4+ CD25+ T cells. Thus, based on the stable immature phenotype of RAPA-DC and their propensity to selectively expand Treg, the combination of co-stimulation blockade with RAPADC administration may prove a highly effective therapy to promote tolerance and thus reduce vascular sclerosis. As such, the current application will define whether alloantiqen (alloAq) Aq presentation by recipient-derived RAPA-DC in combination with blockade of the CD40-CD154 pathway results in lonq-term, rejection-free alloqraft survival through the generation of Treq in a murine heart transplant model. Also, the studies proposed are focused on elucidating the molecular mechanisms that underlie the inhibitory action of RAPA on DC maturation and stimulatory abilitv, and on utilizinq RAPA-DC to identify siqnalinq pathway and qene products that are critical for their expansion of Treq cells. Relevance: The morbidity associated with long-term immunosuppressant administration, and the failure of these treatments to prevent allograft loss due to chronic rejection, remain significant obstacles in clinical solid organ transplantation. Dendritic cells, potent natural regulators of immune responses, if properly utilized as tolerogenic vectors may hold the key to consistent induction of operational tolerance reducing the current dependence on chronic immunosuppression and blocking immunological components of chronic rejection. [unreadable] [unreadable] [unreadable]